Hypoxia (deficiency in oxygen supply) is tightly associated with tumorigenesis and angiogenesis, as well as with cerebral and myocardial ischemia. When challenged by low oxygen tension, cells activate hypoxia-inducible factor 1 (HIF-1), a master regulator of oxygen homeostasis, bringing about transcriptional up-regulation of a variety of hypoxia-responsive genes, including vascular endothelial growth factor, nitric oxide synthases, glucose transporters, and glycolytic enzymes. HIF-1 is a heterodimeric transcription factor, composed of HIF-1a and HIF-1b of the basic helix-loop-helix PAS family. Our research focuses on the molecular mechanisms underlying HIF-1 activation, the role of HIF-1a in tumorigenesis, and the molecular basis for therapeutic drug targeting. Previously, we and others demonstrated that HIF-1 activation is regulated primarily by posttranslational modifications of HIF-1a, resulting in increased protein stability and transcriptional activity. The HIF-1a level is controlled by oxygen-dependent proteolysis via the ubiquitin-proteasome pathway that targets the oxygen-dependent degradation domain (ODD) of HIF-1a. The HIF-1a E3 ubiquitin ligase contains the tumor suppressor von Hippel-Lindau (VHL) protein, which binds ODD and catalyzes polyubiquitination of HIF-1a. The VHL protein interacts with HIF-1a via a specific recognition of hydroxylated Pro402 or Pro564, both of which are modified in normoxia by a family of prolyl-4-hydroxylases functioning as oxygen sensors. To gain insights into the molecular determinant of prolyl hydroxylation, we recently identified a leucine residue (Leu-574) downstream of Pro-564 to be crucial for the VHL-mediated degradation of HIF-1a. Moreover, we have demonstrated that the leucine residue is required for recruiting a key HIF prolyl-4-hydrxylase. Thus, the identification of Leu-574 may provide a molecular basis for drug targeting of HIF-1a activity. Although hypoxia induces an increase in HIF-1a levels, what is equally interesting is why HIF-1a is constitutively degraded. We are in the process of identifying novel functions and regulatory mechanisms of HIF-1a. Solid tumors contain hypoxic regions that often acquire diminished apoptotic potential and resistance to chemo- or radio-therapy. Hypoxia may induce mutations of critical genes, ultimately resulting in genomic instability. The role of HIF-1a in tumorigenesis has been implicated by the heightened expression of HIF-1a and HIF-2a, a close member of the family, and by the correlation of their overexpression with poor diagnosis. Experimental evidence have shown that HIF-1a is required for tumor angiogenesis and hypoxia-induced growth arrest as well. By taking advantage of the HIF-1a variants with defined biochemical activities, we are making progress of characterizing the role of HIF-1a in tumorigenesis, and elucidating the molecular mechanisms underlying hypoxic effects on tumor development. Novel hypotheses have been generated from the study, possibly leading to the invention of new therapeutic drug targets.